Endothelial cells form a semi-permeable barrier that limits fluid, solute, and macromolecular permeability Among pulmonary endothelial cell phenotypes, microvascular endothelial cells (PMVECs) possess the most restrictive barrier, which is necessary to prevent alveolar edema and optimize gas exchange. Junctional integrity is dynamically adjusted by near-membrane cAMP signals, where increased cAMP concentrations promote junctional strength; accordingly, transmembrane adenylyl cyclase activity and near membrane cAMP signals are higher in PMVECs than in pulmonary artery endothelial cells. Pathogenic bacteria, such as Pseudomonas aeruginosa, utilize a soluble adenylyl cyclase toxin, exotoxin Y (ExoY), to produce cAMP within the bulk cytosol, where the cytosolic cAMP signal disassembles microtubules and induces inter- endothelial cell gap formation. Microtubules are stabilized by a microtubule binding protein(s), non-neuronal tau. Non-neuronal tau possesses multiple protein kinase A phosphorylation sites. However, preliminary data reveal that ExoY is a promiscuous cyclase, generating both cytosolic cAMP and cGMP signals that result in non-neuronal tau phosphorylation on serine 214. Tau serine 214 phosphorylation coincides with microtubule disassembly, reminiscent of tau hyperphosphorylation in Alzheimer's disease and related tauopathies, where tau hyperphosphorylation dissociates it from microtubules leading to microtubule disassembly and tau aggregation. Tau is phosphorylated by multiple protein kinases. It is not clear whether ExoY activates protein kinase A and protein kinase G, whether protein kinase A activation is sufficient for tau serine 214 phosphorylation, or whether tau serine 214 phosphorylation is the critical stimulus for microtubule disassembly; indeed, the molecular nature of this endothelial cell tauopathy remains poorly understood Hence, this proposal tests the overall hypothesis that ExoY produces cAMP that is sufficient to activate protein kinase A which induces tau serine 214 phosphorylation, disassembles microtubules, aggregates tau induces intercellular gaps, and increases endothelial cell permeability. Specific aims test the related hypotheses that: [1] ExoY generates cAMP and cGMP that activates protein kinase A and protein kinase G, respectively; [2] Protein kinase A activation is sufficient to cause non-neuronal tau serine 214 phosphorylation that disassembles microtubules; and [3] Non-neuronal tau serine 214 phosphorylation induces endothelial cell gaps and increases permeability. Completion of this work will contribute to our understanding of how cAMP and cGMP signals are compartmentalized in endothelium to dynamically adjust barrier integrity, and further, how the P. aeruginosa ExoY exploits this cellular biology as a pathogenic mechanism of toxicity. RELEVANCE (See instructions): Oxygen delivery from the air to the blood occurs across the lung's alveolar capillary membrane. Capillaries are the smallest blood vessels in the body, and are comprised of a single endothelial cell layer. Disruption of the continuity of this cell layer causes fluid accumulation in the airspace, which prevents oxygen delivery to the blood. Our studies examine ways in which bacteria disrupt the capillary endothelial cell barrier.